We have worked on the RCAS vector system for more than 20 years; we began to use HIV-1 vectors as tools to study HIV-1 replication about 3 years ago. The RCAS vectors are used for two different types of experiments: 1) To express genes in cells in culture or in animal models (both chicken and mouse) to study gene function and the role(s) particular genes play in development and/or pathogenesis; and 2) to study viral replication. We have developed (and continue to develop) the RCAS vectors and model systems to study the effects of gene expression on pathogenesis and development; these are all collaborative efforts. We are continuing to use the RCAS system to study viral replication. The vectors are replication competent, grow to high titer, and are safe and easy to use. However, given the importance of HIV-1 in human disease and the wealth of information about the structure and function of the HIV-1 proteins including reverse transcriptase (RT), we want to understand HIV-1 replication. We are attempting to correlate the structure and function of HIV-1 RT with the process of reverse transcription in cells infected with a one-round HIV-1 vector. Using this system, we can monitor several of the important steps in reverse transcription and the specificity of ribonuclease H (RNase H) cleavage. This is an important aspect of our current and future research; fortunately, many of the lessons we have learned in developing the RCAS vectors are useful in planning the HIV-1 vector experiments, and some of the tools we have developed (in particular, the shuttle cassette from the RCAS-derived RSVP vectors) can, with some modifications, be used in the HIV-1 system. We believe that the interactions between HIV-1 RT and the different types of nucleic acid substrates it uses are important for the ability of the enzyme to constrain, bend, and properly position the nucleic acid substrates. In turn, the proper positioning of the nucleic acid substrates is critically important both for polymerase function and accurate RNase H cleavage. As a consequence, we are particularly interested in amino acids that contact the nucleic acid substrates and their roles in reverse transcription. We have used a combined structural, biochemical, and vector-based approach to analyze the importance of amino acids in the RNase H domain that contact the RNA/DNA substrate. We plan to continue this type of approach with amino acids that contact the nucleic acid and in the polymerase domain, and have started to use similar approaches to analyze the polypurine tract (ppt). We also plan to select pseudorevertants (compensatory mutations) for some of the mutations that have interesting in vivo phenotypes. As has already been discussed, whenever we obtain unexpected phenotypes in vivo, we will use biochemical (and, where appropriate, structural) analysis to try to understand the basis of the in vivo data. Corresponds to Hughes Project 2 in the May 2003 site visit report of the HIV Drug Resistance Program